Methods and compositions for live attenuated viruses

ABSTRACT

Embodiments herein relate to compositions of and methods for live viruses. In certain embodiments, a live, attenuated virus composition includes, but is not limited to, one or more live, attenuated viruses and compositions to reduce inactivation and/or degradation of the live, attenuated virus. In other embodiments, the live, attenuated virus composition may be a vaccine composition. In yet other compositions, a live, attenuated virus composition may include at least one carbohydrate, at least one protein and at least one high molecular weight surfactants for reducing inactivation and/or degradation of the live, attenuated virus.

PRIORITY

This application claims priority under 35 U.S.C. 121 as a divisionalapplication of U.S. patent application Ser. No. 13/300,217, filed Nov.18, 2011, which claims priority under 35 U.S.C. 121 as a divisionalapplication of U.S. patent application Ser. No. 12/098,077, filed Apr.4, 2008, and issued on Dec. 27, 2011, as U.S. Pat. No. 8,084,039, whichclaims the benefit under 35 USC § 119(e) of provisional U.S. PatentApplication Ser. No. 60/910,579 filed on Apr. 6, 2007. Theseapplications are incorporated herein in their entirety for all purposes.

FEDERALLY FUNDED RESEARCH

This invention was made with Government support under U54 AI06537 andU01 AI070443 awarded by the National Institutes of Health. TheGovernment has certain rights in this invention.

FIELD

Embodiments herein relate to compositions and methods for stabilizinglive, attenuated viruses. Other embodiments relate to compositions andmethods for reducing degradation of live, attenuated viruses. Stillother embodiments relate to uses of these compositions in kits forportable applications and methods.

BACKGROUND

Vaccines to protect against viral infections have been effectively usedto reduce the incidence of human disease. One of the most successfultechnologies for viral vaccines is to immunize animals or humans with aweakened or attenuated strain of the virus (a “live, attenuated virus”).Due to limited replication after immunization, the attenuated straindoes not cause disease. However, the limited viral replication issufficient to express the full repertoire of viral antigens andgenerates potent and long-lasting immune responses to the virus. Thus,upon subsequent exposure to a pathogenic strain of the virus, theimmunized individual is protected from disease. These live, attenuatedviral vaccines are among the most successful vaccines used in publichealth.

Ten of the sixteen viral vaccines approved for sale in the U.S. arelive, attenuated viruses. Highly successful live viral vaccines includethe yellow fever 17D virus, Sabin poliovirus types 1, 2 and 3, measles,mumps, rubella, varicella and vaccinia viruses. Use of the vacciniavirus vaccine to control smallpox outbreaks led to the first and onlyeradication of a human disease. The Sabin poliovirus vaccine has helpedprevent crippling disease throughout the world and is being used in theefforts to eradicate polio. Childhood vaccination with measles, mumps,rubella and varicella vaccines prevent millions of deaths and illnessesinternationally.

Recent technical advances, such as reassortment, reverse genetics andcold adaptation, have led to the licensure of live, attenuated virusesfor influenza and rotavirus. A number of live, viral vaccines developedwith recombinant DNA technologies are in human clinical testing,including vaccines for West Nile disease, dengue fever, malaria,tuberculosis and HIV. These recombinant viral vaccines rely onmanipulation of well-characterized attenuated viral vaccines, such asadenovirus, vaccinia virus, yellow fever 17D or the dengue virus, DEN-2PDK-53. The safe, attenuated viruses are genetically engineered toexpress protective antigens for other viral or bacterial pathogens.Several recombinant viral vaccines have been approved for animal use,including a canarypox/feline leukemia recombinant virus, acanarypox/canine distemper recombinant virus, a canarypox/West Nilerecombinant virus and a yellow fever/West Nile recombinant virus. As agroup, the live attenuated virus vaccines are amongst the mostsuccessful medical interventions in human history, second only to theadvent of antibiotics and hold the promise to improve public healththroughout the world.

In order for live, attenuated viral vaccines to be effective, they mustbe capable of replicating after immunization. Thus, any factors thatinactivate the virus can cripple the vaccine. For example, widespreaddistribution and use of the smallpox vaccine prior to World War II waslimited because the virus was inactivated after only a few days atambient temperatures. In the 1920s, French scientists demonstration thatfreeze-dried vaccine provided long term stability and techniques forlarge-scale manufacture of freeze-dried vaccine were developed in the1940s (see for example Collier 1955). In addition to freeze-drying,various additives have been identified that can help stabilize theviruses in live, attenuated viral vaccines (See for example Burke, Hsuet al 1999). These stabilizers typically include one or more of thefollowing components: divalent cations, buffered salt solutions,chelators, urea, sugars (e.g. sucrose, lactose, trehalose), polyols(e.g., glycerol, mannitol, sorbitol, polyethylene glycol), amino acids,protein hydrolystates (e.g. casein hydrolysate, lactalbumin hydrolysate,peptone), proteins (e.g. gelatin, human serum albumin) or polymers (e.g.dextran).

However, even with these stabilizing agents, many of the commonly usedvaccines still require refrigeration for stabilization. Other commonlyused vaccines are sensitive to temperature extremes; either excessiveheat or accidental freezing can inactivate the vaccine. Maintaining this“cold chain” throughout distribution is particularly difficult in thedeveloping world. Thus, there remains a need for improving the stabilityof both existing and newly developed live, attenuated viral vaccines.

Flaviviruses are amongst the most labile viruses. They are envelopedviruses with a RNA genome of approximately 11,000 bases. Most of theflaviviruses are transmitted by an arthropod vector, commonlymosquitoes. There are over 70 different flaviviruses that are groupedinto three major categories based on serology: the dengue group, theJapanese encephalitis group and the yellow fever group. Amongst theknown flaviviruses, 40 are transmitted by mosquitoes, 16 are transmittedby ticks and 18 viruses have no identified insect vector. Thus, mostflaviviruses have evolved to replicate in both their arthropod vectorand their vertebrate host species (often birds or mammals). Expandingurbanization, worldwide travel and environmental changes (such asdeforestation or rain patterns) have lead to the emergence of severalflaviviruses as threats to human public health. Such viruses include,but are not limited to, yellow fever virus, the dengue viruses, WestNile virus, Japanese encephalitis virus, and tick-borne encephalitisviruses.

Through intensive mosquito control and vaccination efforts, yellow feverwas eliminated from much of North, Central and South America, theCaribbean and Europe. However, in the last 20 years, the number ofcountries reporting cases has increased. Yellow fever virus is nowendemic in major portions of Africa and South America and some Caribbeanislands. The World Health Organization (WHO) estimates that 200,000cases of yellow fever occur annually leading to 30,000 deaths. SinceWorld War II, dengue flaviviruses have spread to tropical andsubtropical regions throughout the world and now threaten over 3.5billion people, about half of the world's population. The WHO estimatesthat 50-100 million cases of dengue fever occur annually. 500,000 ofthese are the more severe, life-threatening form of the disease, termeddengue hemorrhagic fever, that leads to more than 25,000 deaths peryear. A particularly virulent form of West Nile virus was introducedinto the Western hemisphere, presumably by travel, in New York in 1999.The mosquito-transmitted virus infected birds as the primary host, butalso caused disease and mortality in humans and horses. West Nile virusspread throughout the United States and into Canada and Mexico. Sinceits introduction, West Nile virus has caused over 20,000 reported casesof West Nile disease leading to 950 deaths in the United States.Japanese encephalitis virus causes 30,000 to 50,000 cases ofneurological disease annually, primarily in eastern and southern Asia.25-30% of the reported cases are fatal. The tick-borne encephalitisviruses are endemic to parts of Europe and Asia and continue to causeepisodic outbreaks affecting thousands of individuals. Related viruseswith more limited geographical spread include Kunjin virus (a closerelative of West Nile) and Murray Valley encephalitis virus in Australiaand New Guinea, St. Louis encephalitis virus in North and South America,the Usutu, Koutango, and Yaonde viruses in Africa, and Cacipacore virusin South American.

Live, attenuated viral vaccines have been developed that are safe andprotect against flavivirus diseases, such as yellow fever and Japaneseencephalitis. The live, attenuated viral vaccine, 17D, has been widelyused to prevent yellow fever. The current flavivirus vaccines arelyophilized in the presence of stabilizers. Nonetheless, the vaccinesrequire storage and shipment at 2-8° C., a requirement that is difficultto achieve in the developing world and more remote regions of developednations. Furthermore, upon reconstitution, the vaccines rapidly losepotency even when stored at 2-8° C.

The measles vaccine is another example of a labile attenuated virus thatis used worldwide to prevent disease. Measles virus is an enveloped,non-segmented negative strand RNA virus of the Paramyxovirus family.Measles is a highly contagious, seasonal disease that can affectvirtually every child before puberty in the absence of vaccination. Indeveloping countries, mortality rates in measles-infected children canbe as high as 2 to 15%. Indeed, despite efforts to institute worldwideimmunization, measles still causes greater than 7,000 deaths in childrenper year. The measles vaccine is a live, attenuated virus that ismanufactured in primary chicken fibroblast cells. The vaccine isstabilized with gelatin and sorbitol and is then lyophilized. Thestabilized, lyophilized vaccine has a shelf life of 2 years or more ifstored at 2 to 8° C. However, the lyophilized vaccine still requires acold chain that is difficult to maintain in the developing world.Furthermore, upon reconstitution, the vaccine loses 50% of its potencywithin 1 hour at room temperature (20 to 25° C.).

Thus, a need exists in the art for improved vaccine formulations.

SUMMARY

Embodiments herein concern methods and compositions to reduce or preventdeterioration or inactivation of a live attenuated virus composition.Certain compositions disclosed can include combinations of componentsthat reduce deterioration of a live attenuated virus. Other embodimentsherein concern combinations of excipients that greatly enhance thestability of live attenuated viruses. Yet other compositions and methodsherein are directed to reducing the need for lower temperatures (e.g.refrigerated or frozen storage) while increasing the shelf life ofaqueous and/or reconstituted live attenuated virus.

In accordance with these embodiments, certain live attenuated virusesare directed to flaviviruses. Some embodiments, directed tocompositions, can include, but are not limited to, one or more live,attenuated viruses, such as one or more live, attenuated flaviviruses incombination with one or more high molecular weight surfactants,proteins, and carbohydrates.

Compositions contemplated herein can increase the stabilization and/orreduce the inactivation and/or degradation of a live attenuated virusincluding, but not limited to, a live attenuated Flavivirus, Togavirus,Coronavirus, Rhabdovirus, Filovirus, Paramyxovirus, Orthomyxovirus,Bunyavirus, Arenavirus, Retrovirus, Hepadnavirus, Pestivirus,Picornavirus, Calicivirus, Reovirus, Parvovirus, Papovavirus,Adenovirus, Herpes virus, or Poxvirus.

Other embodiments concern live, attenuated virus compositions andmethods directed to a vaccine compositions capable of reducing orpreventing onset of a medical condition caused by one or more of theviruses contemplated herein. In accordance with these embodiments,medical conditions may include, but are not limited to, West Nileinfection, dengue fever, Japanese encephalitis, Kyasanur forest disease,Murray valley encephalitis, Alkhurma hemorrhagic fever, St. Louisencephalitis, tick-borne encephalitis, yellow fever and hepatitis Cvirus infection.

In certain embodiments, compositions contemplated herein can bepartially or wholly dehydrated or hydrated. In other embodiments,protein agents contemplated of use in compositions herein can include,but are not limited to, lactalbumin, human serum albumin, a recombinanthuman serum albumin (rHSA), bovine serum albumin (BSA), other serumalbumins or albumin gene family members. Saccharides or polyol agentscan include, but are not limited to, monosaccharides, disaccharides,sugar alcohols, trehalose, sucrose, maltose, isomaltose, cellibiose,gentiobiose, laminaribose, xylobiose, mannobiose, lactose, fructose,sorbitol, mannitol, lactitol, xylitol, erythritol, raffinose, amylase,cyclodextrins, chitosan, or cellulose. In certain embodiments,surfactant agents can include, but are not limited to, a nonionicsurfactant such as alkyl poly(ethylene oxide), copolymers ofpoly(ethylene oxide) and poly(propylene oxide) (EO-PO block copolymers),poly(vinylpyrrolidone), alkyl polyglucosides (such as sucrosemonostearate, lauryl diglucoside, or sorbitan monolaureate, octylglucoside and decyl maltoside), fatty alcohols (cetyl alcohol or olelylalcohol), or cocamides (cocamide MEA, cocamide DEA and cocamide TEA).

In other embodiments, the surfactants can include, but are not limitedto, copolymer poloxamer 407 (Pluronic F127®), poloxamer 188 (PluronicF68®), poloxamer 403 (Pluronic P123®), or other EO-PO block copolymersof greater than 3,000-4,000 MW.

In some embodiments, vaccine compositions can include, but are notlimited to, one or more protein agent that is serum albumin; one or moresaccharide agent that is trehalose; and one or more surfactant polymeragent that is the EO-PO block copolymer poloxamer 407 (Pluronic F127®).

Some embodiments herein concern partially or wholly dehydrated live,attenuated viral compositions. In accordance with these embodiments, acomposition may be 20% or more; 30% or more; 40% or more; 50% or more;60% or more; 70% or more; 80% or more; or 90% or more dehydrated.

Other embodiments concern methods for decreasing inactivation of a liveattenuated viruses including, but not limited to, combining one or morelive attenuated viruses with a composition capable of reducinginactivation of a live, attenuated virus including, but not limited to,one or more protein agents; one or more saccharides or polyols agents;and one or more high molecular weight surfactants, wherein thecomposition decreases inactivation of the live attenuated virus. Inaccordance with these embodiments, the live attenuated virus mayinclude, but is not limited to, a Flavivirus, Togavirus, Coronavirus,Rhabdovirus, Filovirus, Paramyxovirus, Orthomyxovirus, Bunyavirus,Arenavirus, Retrovirus, Hepadnavirus, Pestivirus, Picornavirus,Calicivirus, Reovirus, Parvovirus, Papovavirus, Adenovirus, Herpesvirus, or a Poxvirus. Additionally, methods and compositions disclosedherein can include freeze drying or other dehydrating methods for thecombination. In accordance with these methods and compositions, themethods and compositions decrease inactivation of the freeze dried orpartially or wholly dehydrated live attenuated virus. In other methods,compositions for decreasing inactivation of a live attenuated virus maycomprise an aqueous composition or may comprise a rehydrated compositionafter dehydration. Compositions described herein are capable ofincreasing the shelf life of an aqueous or rehydrated live attenuatedvirus.

In certain particular embodiments, a live attenuated virus for use in avaccine composition contemplated herein may include, but is not limitedto, one or more live, attenuated flavivirus vaccines, including but notlimited to, attenuated yellow fever viruses (such as 17D), attenuatedJapanese encephalitis viruses, (such as SA 14-14-2), attenuated dengueviruses (such as DEN-2/PDK-53 or DEN-4Δ30) or recombinant chimericflaviviruses.

In certain embodiments, compositions contemplated herein are capable ofdecreasing inactivation and/or degradation of a hydrated live attenuatedvirus for greater than 24 hours at room temperatures (e.g. about 20° toabout 25° C.) or refrigeration temperatures (e.g. about 0° to about 10°C.). In more particular embodiments, a combination composition iscapable of maintaining about 100 percent of the live attenuated virusfor greater than 24 hours. In addition, combination compositionscontemplated herein are capable of reducing inactivation of a hydratedlive attenuated virus during at least 2 freeze and thaw cycles. Othermethods concern combination compositions capable of reducinginactivation of a hydrated live attenuated virus for about 24 hours toabout 50 days at refrigeration temperatures (e.g. about 0° to about 10°C.). Compositions contemplated in these methods, can include, but arenot limited to, one or more protein agent of serum albumin; one or moresaccharide agent of trehalose; and one or more EO-PO block copolymeragent of copolymer poloxamer 407 (Pluronic F127®). In certainembodiments, the live, attenuated virus composition remains at about100% viral titer after 7 days at approximately 21° C. and about 100%viral titer after 50 days at refrigeration temperatures around 4° C.Other embodiments herein may include live, attenuated virus compositionremaining at about 90%, or about 80% viral titer after 7 days atapproximately 21° C. and about 90%, or about 80% viral titer after 50days at refrigeration temperatures around 4° C. Other embodimentscontemplated include live, attenuated virus compositions remaining atabout 3× to about 10× the concentration of viral titer after severalhours (e.g. 20 hours) at approximately 37° C. compared to othercompositions known in the art. (see for example, FIGS. 4 and 5).Compositions disclosed herein reduce degradation of the live, attenuatedvirus when the composition is stored at approximately 37° C.

Other embodiments concern kits for decreasing the inactivation of alive, attenuated virus composition including, but not limited to, acontainer; and a composition including, but not limited to, one or moreprotein agents, one or more saccharide or polyol agents, and one or moreEO-PO block copolymer agents, wherein the composition decreasesinactivation and/or degradation of a live, attenuated virus. Inaccordance with these embodiments, a kit composition may include one ormore protein agents of serum albumin; one or more saccharide agent oftrehalose; and one or more EO-PO block copolymer agent. Additionally, akit contemplated herein may further include one or more live, attenuatedviruses including, but not limited to, a Flavivirus, Togavirus,Coronavirus, Rhabdovirus, Filovirus, Paramyxovirus, Orthomyxovirus,Bunyavirus, Arenavirus, Retrovirus, Hepadnavirus, Pestivirus,Picornavirus, Calicivirus, Reovirus, Parvovirus, Papovavirus,Adenovirus, Herpes virus, or Poxvirus. In certain embodiments,compositions herein can include trehalose as a saccharide agent. Inaccordance with these embodiments, trehalose concentration may be equalto or greater than 5% (w/v). In certain embodiments, compositions hereincan include poloxamer 407 (Pluronic F127®) as an EO-PO block copolymeragent. In accordance with these embodiments, poloxamer 407 (PluronicF127®) concentration may be about 0.1 to about 4 percent (w/v).

In other embodiments, compositions contemplated herein may contain traceamounts or no divalent cations. For example, compositions contemplatedherein may have trace amounts or no calcium/magnesium (Ca⁺²/Mg⁺²).

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the instant specification and areincluded to further demonstrate certain aspects of particularembodiments herein. The embodiments may be better understood byreference to one or more of these drawings in combination with thedetailed description presented herein.

FIG. 1 represents an exemplary histogram of experiments using variouscompositions for testing the stability of an exemplary virus, DEN-2 PDK53 flavivirus, in the compositions.

FIG. 2 represents an exemplary graph of a kinetic analysis of anexemplary virus, DEN-2 PDK 53 flavivirus, for viral inactivation at 37°C. in various exemplary compositions.

FIG. 3 represents an exemplary histogram of an analysis of an exemplaryvirus, DEN-2 PDK 53 virus, stored at 37° C. for 21 hours. Values areexpressed as a percentage of the viral titer remaining after incubationrelative to the input titer. Formulation percentages refer to (w/v) ofthe respective excipient.

FIG. 4 represents an exemplary histogram of an analysis of an exemplaryvirus, DEN-2 PDK 53 virus, stored at 37° C. for 23 hours in differentcompositions. Values are expressed as a percentage of the viral titerremaining after incubation relative to the input titer.

FIG. 5 represents an exemplary histogram of an analysis of an exemplaryvirus, DEN-2 PDK 53 virus, stored at 37° C. for 23 hours in differentcompositions. Values are expressed as a percentage of the viral titerremaining after incubation relative to the input titer. The two bars foreach formulation represent duplicates in the experiment.

FIG. 6 represents an exemplary histogram analysis of an exemplary virus,DEN-2 PDK 53 virus, after two freeze-thaw cycles when stored indifferent formulations. Values are expressed as a percentage of theviral titer remaining after freeze-thaw cycles relative to the inputtiter.

FIG. 7 represents an exemplary graph of a kinetic analysis of anexemplary virus, DEN-2 PDK 53/WN recombinant flavivirus, in variousexemplary compositions for viral inactivation at 25° C. over severalweeks of time.

FIG. 8 represents an exemplary graph of a kinetic analysis of anexemplary virus, DEN-2 PDK 53/WN recombinant flavivirus, in variousexemplary compositions for viral inactivation at 4° C. over severalweeks of time.

FIG. 9 represents an exemplary histogram analysis of an exemplary virus,DEN-2 PDK-53 virus, after lyophilization in various exemplarycompositions. Viral inactivation was assessed as described above aftertwo weeks at different temperatures.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Definitions

As used herein, “a” or “an” may mean one or more than one of an item.

As used herein, “about” may mean up to and including plus or minus fivepercent, for example, about 100 may mean 95 and up to 105.

As used herein, “saccharide” agents can mean one or moremonosaccharides, (e.g. glucose, galactose, ribose, mannose, rhamnose,talose, xylose, or allose arabinose), one or more disaccharides (e.g.trehalose, sucrose, maltose, isomaltose, cellibiose, gentiobiose,laminaribose, xylobiose, mannobiose, lactose, or fructose),trisaccharides (e.g. acarbose, raffinose, melizitose, panose, orcellotriose) or sugar polymers (e.g. dextran, xanthan, pullulan,cyclodextrins, amylose, amylopectin, starch, cellooligosaccharides,cellulose, maltooligosaccharides, glycogen, chitosan, or chitin).

As used herein, “polyol” agents can mean any sugar alcohol (e.g.mannitol, sorbitol, arabitol, erythritol, maltitol, xylitol, glycitol,glycol, polyglycitol, polyethylene glycol, polypropylene glycol, orglycerol). As used herein, “high molecular weight surfactants” can meana surface active, amphiphilic molecule greater than 1500 molecularweight.

As used herein, “EO-PO block copolymer” can mean a copolymer consistingof blocks of poly(ethylene oxide) poly(propylene oxide). In addition, asused herein, “Pluronic” can mean EO-PO block copolymers in theEOx-POy-EOx. This configuration of EO-PO block copolymer is alsoreferred to as “Poloxamer” or “Synperonic”.

As used herein, “attenuated virus” can mean a virus that demonstratesreduced or no clinical signs of disease when administered to an animal.

DETAILED DESCRIPTIONS

In the following sections, various exemplary compositions and methodsare described in order to detail various embodiments. It will be obviousto one skilled in the art that practicing the various embodiments doesnot require the employment of all or even some of the specific detailsoutlined herein, but rather that concentrations, times and otherspecific details may be modified through routine experimentation. Insome cases, well known methods or components have not been included inthe description.

Stability of flavivirus vaccines has been assessed for both the existingyellow fever and Japanese encephalitis live, attenuated viruses. Whentested in 1987, only five of the twelve yellow fever vaccinesmanufactured at that time met minimal standards of stability.Subsequently, addition of a mixture of sugars, amino acids and divalentcations was demonstrated to stabilize the lyophilized vaccine, so thatthe vaccine lost less than 1 log of potency after incubation at 37° C.for 14 days. Stabilizing lyophilized formulations for the yellow fevervaccine have been described (see for example U.S. Pat. No. 4,500,512).U.S. Pat. No. 4,500,512, describes a combination of lactose, sorbitol,the divalent cations, calcium and magnesium, and at least one aminoacid. While this formulation may help to stabilize the lyophilizedvaccine, it fails to provide stability to the vaccine in aqueous form.Another study examined the ability of several different formulationsincluding the compositions described above and the effect of sucrose,trehalose and lactalbumin on the stability of the lyophilized yellowfever vaccine. Formulations consisting of 10% sucrose alone, 2% sorbitolwith 4% inositol, or 10% sucrose with 5% lactalbumin, 0.1 g/l CaCl2 and0.076 g/l MgSO4 were found to provide the best stability (see forexample Adebayo, Sim-Brandenburg et al. 1998). However, in all casesafter resuspension, yellow fever vaccine is still very unstable and mustbe discarded after only about one hour (see for example Monath 1996;Adebayo, Sim-Brandenburg et al. 1998). This leads to vaccine wastage andthe potential to cause administration of ineffective vaccine under fieldconditions, if an unstable vaccine is used.

Another live, attenuated flavivirus vaccine for protection againstJapanese encephalitis has been licensed and is in widespread use inChina (see for example Halstead and Tsai 2004). The Japaneseencephalitis vaccine strain, SA 14-14-2, is grown on primary hamsterkidney cells and the cell supernatant is harvested and coarselyfiltered. One previous composition included 1% gelatin and 5% sorbitoladded as stabilizers. Using these stabilizers, the vaccine islyophilized and then is stable at 2 to 8° C. for at least 1.5 years, butfor only 4 months at room temperature and 10 days at 37° C. As with theyellow fever vaccine, the reconstituted vaccine is very labile and isstable for only 2 hours at room temperature (see for example Wanf, Yanget al 1990). In certain embodiments herein, live, attenuated flaviviruscompositions for stabilizing or reducing the degradation of Japaneseencephalitis are contemplated.

No formulation for a live, attenuated flavivirus vaccine has beenidentified that provides long term stability of lyophilized formulationsat temperatures greater than 2-8° C. In addition, no formulation hasbeen described that prevents loss of titer, stabilizes or reducesdegradation of aqueous vaccines for greater than a few hours.

Formulations for other live, attenuated viruses have also been described(see for example Burke, Hsu et al. 1999). One common stabilizer,referred to as SPGA is a mixture of 2 to 10% sucrose, phosphate,potassium glutamate and 0.5 to 2% serum albumin (see for exampleBovarnick, Miller et al. 1950). Various modifications of this basicformulation have been identified with different cations, withsubstitutions of starch hydrolysate or dextran for sucrose, and withsubstitutions of casein hydrolysate or poly-vinyl pyrrolidone for serumalbumin. Other formulations use hydrolyzed gelatin instead of serumalbumin as a protein source (Burke, Hsu et al 1999). However, gelatincan cause allergic reactions in immunized children and could be a causeof vaccine-related adverse events. U.S. Pat. No. 6,210,683 describes thesubstitution of recombinant human serum albumin for albumin purifiedfrom human serum in vaccine formulations.

Embodiments herein disclose compositions that enhance the stability ofand/or reduce deterioration of live, attenuated virus vaccines comparedto those in the prior art. Certain compositions disclosed herein providestability of aqueous viruses for up to 2 hours; up to 3 hours; up to 4hours and greater than 4 hours at or about 37° C. Certain compositionsdisclosed herein provide stability of aqueous viruses for up to 1 day toabout 1 week or more, at or about room temperature (e.g. 25° C.).Embodiments contemplated herein provide increased protection of a live,attenuated virus from for example, freezing and/or thawing, and/orelevated temperatures. In certain embodiments, compositions herein canstabilize, reduce deterioration and/or prevent inactivation ofdehydrated live, attenuated viral products in room temperatureconditions (e.g. about 25° C.). In other embodiments, compositionscontemplated herein can stabilize, reduce deterioration and/or preventinactivation of aqueous live, attenuated viral products at about 25° C.or up to or about 37° C. Compositions and methods disclosed herein canfacilitate the storage, distribution, delivery and administration ofviral vaccines in developed and under developed regions.

Other embodiments can include compositions for live attenuated virusvaccines including, but not limited to, Picornaviruses (e.g., poliovirus, foot and mouth disease virus), Caliciviruses (e.g., SARS virus,and feline infectious peritonitis virus), Togaviruses (e.g., sindbisvirus, the equine encephalitis viruses, chikungunya virus, rubellavirus, Ross River virus, bovine diarrhea virus, hog cholera virus),Flaviviruses (e.g., dengue virus, West Nile virus, yellow fever virus,Japanese encephalitis virus, St. Louis encephalitis virus, tick-borneencephalitis virus), Coronaviruses (e.g., human coronaviruses (commoncold), swine gastroenteritis virus), Rhabdoviruses (e.g., rabies virus,vesicular stomatitis viruses), Filoviruses (e.g., Marburg virus, Ebolavirus), Paramyxoviruses (e.g., measles virus, canine distemper virus,mumps virus, parainfluenza viruses, respiratory syncytial virus,Newcastle disease virus, rinderpest virus), Orthomyxoviruses (e.g.,human influenza viruses, avian influenza viruses, equine influenzaviruses), Bunyaviruses (e.g., hantavirus, LaCrosse virus, Rift Valleyfever virus), Arenaviruses (e.g., Lassa virus, Machupo virus),Reoviruses (e.g., human reoviruses, human rotavirus), Birnaviruses(e.g., infectious bursal virus, fish pancreatic necrosis virus),Retroviruses (e.g., HIV 1, HIV 2, HTLV-1, HTLV-2, bovine leukemia virus,feline immunodeficiency virus, feline sarcoma virus, mouse mammary tumorvirus), Hepadnaviruses (e.g., hepatitis B virus), Parvoviruses (e.g.,human parvovirus B, canine parvovirus, feline panleukopenia virus)Papovaviruses (e.g., human papillomaviruses, SV40, bovinepapillomaviruses), Adenoviruses (e.g., human adenovirus, canineadenovirus, bovine adenovirus, porcine adenovirus), Herpes viruses(e.g., herpes simplex viruses, varicella-zoster virus, infectious bovinerhinotracheitis virus, human cytomegalovirus, human herpesvirus 6), andPoxviruses (e.g., vaccinia, fowlpoxviruses, raccoon poxvirus, skunkpoxvirus, monkeypoxvirus, cowpox virus, musculum contagiosum virus).

Those skilled in the art will recognize that compositions or formulasherein relate to viruses that are attenuated by any means, including butnot limited to, cell culture passage, reassortment, incorporation ofmutations in infectious clones, reverse genetics, other recombinant DNAor RNA manipulation. In addition, those skilled in the art willrecognize that other embodiments relate to viruses that are engineeredto express any other proteins or RNA including, but not limited to,recombinant flaviviruses, recombinant adenoviruses, recombinantpoxviruses, recombinant retroviruses, recombinant adeno-associatedviruses and recombinant herpes viruses. Such viruses may be used asvaccines for infectious diseases, vaccines to treat oncologicalconditions, or viruses to introduce express proteins or RNA (e.g., genetherapy, antisense therapy, ribozyme therapy or small inhibitory RNAtherapy) to treat disorders.

In some embodiments, compositions herein can contain one or more viruseswith membrane envelopes (e.g., enveloped viruses) of the Togavirus,Flavivirus, Coronavirus, Rhabdovirus, Filovirus, Paramyxovirus,Orthomyxovirus, Bunyavirus, Arenavirus, Retrovirus, Hepadnavirus,Herpesvirus or Poxvirus families. In certain embodiments compositionscontain one or more enveloped RNA viruses of the Togavirus, Flavivirus,Coronavirus, Rhabdovirus, Filovirus, Paramyxovirus, Orthomyxovirus,Bunyavirus, Arenavirus, or Retrovirus families. In other embodiments,compositions herein can contain one or more enveloped, positive strandRNA virus of the Togavirus, Flavivirus, Coronavirus, or Retrovirusfamilies. In certain embodiments, compositions can contain one or morelive, attenuated Flaviviruses (e.g., dengue virus, West Nile virus,yellow fever virus, or Japanese encephalitis virus).

Some embodiments herein relate to compositions for live, attenuatedviruses in aqueous or lyophilized form. Those skilled in the art willrecognize that formulations that improve thermal viral stability andprevent freeze-thaw inactivation will improve products that are liquid,powdered, freeze-dried or lyophilized and prepared by methods known inthe art. After reconstitution, such stabilized vaccines can beadministered by a variety routes, including, but not limited tointradermal administration, subcutaneous administration, intramuscularadministration, intranasal administration, pulmonary administration ororal administration. A variety of devices are known in the art fordelivery of the vaccine including, but not limited to, syringe andneedle injection, bifurcated needle administration, administration byintradermal patches or pumps, needle-free jet delivery, intradermalparticle delivery, or aerosol powder delivery.

Embodiments can include compositions consisting of one or more liveattenuated viruses (as described above) and a mixture of one or morehigh molecular weight surfactants and one or more proteins in aphysiological acceptable buffer. In certain embodiments, compositionsinclude, but are not limited to one or more live attenuated viruses, oneor more high molecular weight surfactants, one or more proteins, and oneor more carbohydrates, in a physiological acceptable buffer.

In other embodiments, compositions can contain one or more highmolecular weight surfactants that increase the thermal stability oflive, attenuated viruses. Surfactants have been incorporated intovaccine formulations to prevent material loss to surfaces such as glassvials (see for example Burke, Hsu et al. 1999). However, certainembodiments herein include high molecular weight surfactants with someunusual biochemical properties of utility for compositions and methodsdisclosed herein. The EO-PO block copolymers can include blocks ofpolyethylene oxide (—CH₂CH₂O— designated EO) and polypropylene oxide(—CH₂CHCH₃O-designated PO). The PO block can be flanked by two EO blocksin a EO_(x)-PO_(y)-EO_(x) arrangement. Since the PO component ishydrophilic and the EO component is hydrophobic, overall hydrophilicity,molecular weight and the surfactant properties can be adjusted byvarying x and y in the EO_(x)-PO_(y)-EO_(x) block structure. In aqueoussolutions, the EO-PO block copolymers will self-assemble into micelleswith a PO core and a corona of hydrophilic EO groups. EO-PO blockcopolymer formulations have been investigated as potential drug deliveryagents for a variety of hydrophobic drugs and for protein, DNA orinactivated vaccines (e.g. Todd, Lee et al. 1998; Kabanov, Lemieux etal. 2002). At high concentrations (for example: >than 10%) certain ofthe higher molecular weight EO-PO block copolymers will undergo reversegelation, forming a gel as the temperature increases. Gel formation atbody temperatures permits use of the EO-PO block copolymer gels to actas a depot in drug and vaccine delivery applications (see for exampleCoeshott, Smithson et al. 2004). In addition, due to their surfactantproperties, these polymers have been used in adjuvant formulations, andas an emulsifier in topically applied creams and gels. The EO-PO blockcopolymers have also been shown to accelerate wound and burn healing andto seal cell membranes after radiation or electroporation-mediateddamage.

In other embodiments, vaccine compositions can include one or moresurfactants with molecular weight of 1500 or greater. In a certainembodiment, the surfactant is a non-ionic, hydrophilic,polyoxyethylene-polyoxypropylene block copolymer (or EO-PO blockcopolymer). While EO-PO block copolymers have been used as adjuvants anddelivery vehicles for inactivated vaccines, protein vaccines or DNAvaccines, their use to prevent inactivation of a live virus is notanticipated in the art. In a particular embodiment, a formulation cancontain one or more EO-PO polymers with a molecular weight of 3,000 orgreater. In further embodiments, compositions can include in part anEO-PO block copolymer Pluronic F127® (poloxamer 407) or Pluronic P123®(poloxamer 403). Those skilled in the art will recognize thatmodifications of the surfactants can be chemically made. It iscontemplated herein any essentially equivalent surfactant polymers areconsidered.

Embodiments herein can include compositions of one or more live,attenuated viruses, one or more surfactants and one or more proteins. Incertain embodiments, a protein can be an albumin. Serum albumin is oneof the most common proteins in vertebrate blood and has multiplefunctions. The protein is 585 amino acids with a molecular weight of66500. Human serum albumin is not glycosylated and has a single freethiol group implicated in some of its myriad binding activities. Serumalbumin is predominantly α-helix with three structural domains, eachsubdivided into two subdomains. Albumin is known to specifically bind avariety of molecules, including drugs such as aspirin, ibuprofen,halothane, propofol and warfarin as well as fatty acids, amino acids,steroids, glutathione, metals, bilirubin, lysolecithin, hematin, andprostaglandins. The different structural domains are implicated in drugbinding; most small molecule drugs and hormones bind to one of twoprimary sites located in subdomains IIA and IIIA. Due to its lack ofimmunogenicity, albumin is commonly used as a carrier protein inbiological products. Since the protein dose contained in a live,attenuated viral vaccine can be fractions of a microgram (derived from10³ to 10⁵ viral particles), an inert carrier protein is used to preventloss due to absorption and non-specific binding to glass, plastic orother surfaces. However, as demonstrated herein, an unexpectedimprovement in stability was observed with the combination of an albuminand EO-PO block copolymers suggesting interactions between the twocomponents and/or between the components and the viral particles. Inaddition, enhanced stabilization of viruses in the presence of albuminis not likely due to function as a general carrier protein: otherproteins such as gelatin and lactoferrin fail to improve virusstability.

In certain embodiments, serum albumin may be from a human or othermammalian source. For vaccines intended for human use, particularembodiments can include human albumin or other human products as neededin order to reduce or eliminate adverse immune responses. Those skilledin the art will recognize that albumins specific for each species may beused in animal vaccines (e.g. canine albumin for canine products, bovinealbumin for bovine products). In further embodiments, the protein is arecombinant human albumin. Standard methods exist for expressingrecombinant human albumin or portions thereof in a variety of expressionsystems including bacteria, yeast, algae, plant, mammalian cell ortransgenic animal systems. In addition, serum albumin or portionsthereof can be produced in cell-free systems or chemically synthesized.Recombinant human albumin produced in these or in any similar system isincorporated herein. Those skilled in the art will recognize that otherproteins can substitute for albumin. For example, albumin is a member ofa multi-gene family. Due to their structural and sequence similarities,other members of the family (e.g. α-fetoprotein, vitamin D bindingprotein, or afamin) may substitute for albumin in compositions andmethods contemplated herein. Those skilled in the art will alsorecognize that modifications can be made to albumin by any means knownin the art, for example, by recombinant DNA technology, bypost-translational modification, by proteolytic cleavage and/or bychemical means. Those substitutions and alterations to albumin thatprovide essentially equivalent stabilizing function to serum albuminwithout substitutions and alterations are contemplated herein.

In certain embodiments, compositions having a high molecular weightsurfactant, a protein and a carbohydrate in a pharmaceuticallyacceptable buffer are described. In some embodiments, the carbohydrateis a sugar or a polyol. Sugars can include, but are not limited to,monosaccharides, (e.g. glucose, galactose, ribose, mannose, rhamnose,talose, xylose or allose arabinose), disaccharides (e.g. trehalose,sucrose, maltose, isomaltose, cellibiose, gentiobiose, laminaribose,xylobiose, mannobiose, lactose, or fructose), trisaccharides (e.g.acarbose, raffinose, melizitose, panose, or cellotriose) or sugarpolymers (e.g. dextran, xanthan, pullulan, cyclodextrins, amylose,amylopectin, starch, celloligosaccharides, cellulose,maltooligosaccharides, glycogen, chitosan, or chitin). Polyols caninclude, but are not limited to, mannitol, sorbitol, arabitol,erythritol, maltitol, xylitol, glycitol, glycol, polyglycitol,polyethylene glycol, polypropylene glycol, and glycerol.

In a particular embodiment, formulations can contain a combination ofone or more EO-PO block copolymers, one or more proteins, and trehalosein a pharmacologically acceptable buffer. In certain embodiments,trehalose can be present at concentrations ranging from 5 to 50% (w/v).Trehalose has been used to enhance the stability of proteinformulations. It is widely known in the art as a cryopreservative and isused in nature to protect organisms from stress. Anhydrobiotic organismsthat can tolerate low water conditions contain large amounts oftrehalose. Trehalose has been shown to prevent both membrane fusionevents and phase transitions that can cause membrane destabilizationduring drying. Structural analysis suggests that trehalose fits wellbetween the polar head groups in lipid bylayers. Trehalose also preventsdenaturation of labile proteins during drying. It is thought thattrehalose stabilizes proteins by hydrogen bonding with polar proteinresidues. Trehalose is a disaccharide consisting of two glucosemolecules in a 1:1 linkage. Due to the 1:1 linkage, trehalose has littleor no reducing power and is thus essentially non-reactive with aminoacids and proteins. This lack of reducing activity may improve thestabilizing effect of trehalose on proteins. In certain embodiments,trehalose provides stability to live, attenuated viruses. This activityof trehalose may be due to its ability to stabilize both the membranesand coat proteins of the viruses.

In further embodiments, compositions can include one or more EO-PO blockcopolymers, one or more proteins and one or more carbohydrates, whereone of the carbohydrates is chitosan, in a physiological acceptablebuffer to provide improved stability to live, attenuated viruses. Incertain embodiments, compositions can include chitosan at concentrationsranging from 0.001 to 2% (e.g at a pH of about 6.8). Chitosan is acationic polysaccharide derived by deacetylation of chitin, thestructural polymer of crustacean exoskeletons. It is a polymer ofN-acetyl-glucosamine and glucosamine; the content of the twocarbohydrates depends on the extent of deacetylation. Chitosan'spositive charge allows it to bind to negatively charged surfaces andmolecules. Thus, it binds mucosal surfaces and is thought to promotemucosal absorption. Chitosan also can bind and form nanoparticles withDNA, RNA and other oligonucleotides and has been used in non-viral genedelivery. Certain embodiments herein demonstrate that chitosan increaseslive, attenuated virus stability.

In certain embodiments, compositions can be described that typicallyinclude a physiologically acceptable buffer. Those skilled in the artrecognize that a variety of physiologically acceptable buffers exist,including, but not limited to buffers containing phosphate, TRIS, MOPS,HEPES, bicarbonate, other buffers known in the art ad combinations ofbuffers. In addition, those skilled in the art recognize that adjustingsalt concentrations to near physiological levels (e.g., saline or 0.15 Mtotal salt) may be optimal for parenteral administration of compositionsto prevent cellular damage and/or pain at the site of injection. Thoseskilled in the art also will recognize that as carbohydrateconcentrations increase, salt concentrations can be decreased tomaintain equivalent osmolarity to the formulation. In certainembodiments, a buffering media with pH greater than 6.8 is contemplated;some live, attenuated viruses (e.g. flaviviruses) are unstable at lowpH. In another embodiment, physiologically acceptable buffer can bephosphate-buffered saline (PBS).

Some live, attenuated viral vaccine compositions herein concerncompositions that increase stability and/or reduce deterioration oflive, attenuated virus in addition to having reduced immunogenicity orare non-immunogenic. In accordance with these embodiments, compositionscan include one or more protein agents; one or more saccharides orpolyols agents; and one or more high molecular weight surfactants,wherein the composition decreases inactivation of the live attenuatedvirus. Therefore, certain compositions contemplated herein have reducedadverse reaction when administered to a subject. In some exemplarycompositions, the surfactant agent(s) consists of one or more EO-POblock copolymers; the protein agent(s) are selected from the groupconsisting of lactalbumin, serum albumin, α-fetoprotein, vitamin Dbinding protein, afamin derived from a vertebrate species; and thecarbohydrate agent(s) is one or more of a saccharide and/or a polyol. Incertain embodiments, compositions can include one or more of thecarbohydrate agent(s) selected from the group consisting of trehalose,sucrose, chitosan, sorbitol, and mannitol. In certain more particularembodiments, in order to reduce immune reaction to a vaccine, the serumalbumin can be derived from a vertebrate species or in otherembodiments, from the same source as the subject (e.g. human). In otherembodiments, the carbohydrate agent is trehalose. In certainembodiments, at least one surfactant agent is the EO-N) block copolymer,poloxamer 407 (Pluronic F127®). In some live, attenuated viral vaccinecompositions at least one carbohydrate agent is trehalose. In certainembodiments, live, attenuated viral vaccine compositions include theEO-PO block copolymer, poloxamer 407 (Pluronic F127®) where theconcentration is from 0.1 to 4% (w/v); and/or serum albuminconcentration from 0.001 to 3% (w/v) and/or the trehalose concentrationcan be from 5 to 50% (w/v).

Pharmaceutical Compositions

Embodiments herein provide for administration of compositions tosubjects in a biologically compatible form suitable for pharmaceuticaladministration in vivo. By “biologically compatible form suitable foradministration in vivo” is meant a form of the active agent (e.g. live,attenuated virus composition of the embodiments) to be administered inwhich any toxic effects are outweighed by the therapeutic effects of theactive agent. Administration of a therapeutically active amount of thetherapeutic compositions is defined as an amount effective, at dosagesand for periods of time necessary to achieve a desired result. Forexample, a therapeutically active amount of a compound may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability formulations to elicit a desiredresponse in the individual. Dosage regimen may be adjusted to providethe optimum therapeutic response.

In some embodiments, composition (e.g. pharmaceutical chemical, protein,peptide of an embodiment) may be administered in a convenient mannersuch as subcutaneous, intravenous, by oral administration, inhalation,transdermal application, intravaginal application, topical application,intranasal or rectal administration. In a more particular embodiment,the compound may be orally or subcutaneously administered. In anotherembodiment, the compound may be administered intravenously. In oneembodiment, the compound may be administered intranasally, such asinhalation.

A compound may be administered to a subject in an appropriate carrier ordiluent, co-administered with the composition. The term“pharmaceutically acceptable carrier” as used herein is intended toinclude diluents such as saline and aqueous buffer solutions. The activeagent may also be administered parenterally or intraperitoneally.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations may contain a preservative toprevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use may beadministered by means known in the art. For example, sterile aqueoussolutions (where water soluble) or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersion may be used. In all cases, the composition can be sterile andcan be fluid to the extent that easy syringability exists. It mayfurther be preserved against the contaminating action of microorganismssuch as bacteria and fungi. The pharmaceutically acceptable carrier canbe a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants.

Sterile injectable solutions can be prepared by incorporating activecompound in an amount with an appropriate solvent or with one or acombination of ingredients enumerated above, as required, followed bysterilization.

Upon formulation, solutions can be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above.It is contemplated that slow release capsules, timed-releasemicroparticles, and the like can also be employed for administeringcompositions herein. These particular aqueous solutions are especiallysuitable for intravenous, intramuscular, subcutaneous andintraperitoneal administration.

The active therapeutic agents may be formulated within a mixture caninclude about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1milligrams, or about 0.1 to 1.0 or even about 1 to 10 gram per dose.Single dose or multiple doses can also be administered on an appropriateschedule for a predetermined situation. In some embodiments, doses canbe administered before, during and/or after exposure to a viruscontemplated herein.

In another embodiment, nasal solutions or sprays, aerosols or inhalantsmay be used to deliver the compound of interest. Additional formulationsthat are suitable for other modes of administration includesuppositories and pessaries. A rectal pessary or suppository may also beused. In general, for suppositories, traditional binders and carriersmay include, for example, polyalkylene glycols or triglycerides; suchsuppositories may be formed from mixtures containing the activeingredient in the range of 0.5% to 10%, preferably 1% to 2%.

Oral formulations include such normally employed excipients as, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate and thelike. In certain embodiments, oral pharmaceutical compositions caninclude an inert diluent or assimilable edible carrier, or may beenclosed in hard or soft shell gelatin capsule, or may be compressedinto tablets, or may be incorporated directly with the food of the diet.For oral therapeutic administration, the active compounds may beincorporated with excipients and used in the form of ingestible tablets,buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. Such compositions and preparations should contain at least0.1% of active compound. The percentage of the compositions andpreparations may, of course, be varied and may conveniently be betweenabout 2 to about 75% of the weight of the unit, or preferably between25-60%. The amount of active compounds in such therapeutically usefulcompositions is such that a suitable dosage can be obtained.

Kits

Further embodiments concerns kits for use with methods and compositionsdescribed herein. Compositions and live virus formulations may beprovided in the kit. The kits can also include a suitable container,live, attenuated virus compositions detailed herein and optionally oneor more additional agents such as other anti-viral agents, anti-fungalor anti-bacterial agents.

The kits may further include a suitably aliquoted composition of use ina subject in need thereof. In addition, compositions herein may bepartially or wholly dehydrated or aqueous. Kits contemplated herein maybe stored at room temperatures or at refrigerated temperatures asdisclosed herein depending on the particular formulation.

The container means of the kits will generally include at least onevial, test tube, flask, bottle, syringe or other container means, intowhich a composition may be placed, and preferably, suitably aliquoted.Where an additional component is provided, the kit will also generallycontain one or more additional containers into which this agent orcomponent may be placed. Kits herein will also typically include a meansfor containing the agent, composition and any other reagent containersin close confinement for commercial sale. Such containers may includeinjection or blow-molded plastic containers into which the desired vialsare retained.

EXAMPLES

The following examples are included to demonstrate certain embodimentspresented herein. It should be appreciated by those of skill in the artthat the techniques disclosed in the Examples which follow representtechniques discovered to function well in the practices disclosedherein, and thus can be considered to constitute preferred modes for itspractice. However, those of skill in the art should, in light of thepresent disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope herein.

Example 1 Base Stability of DEN-2 PDK 53 Flavivirus in Liquid Phase

In one illustrative method, the thermal stability for flaviviruses inliquid phase was investigated. In accordance with this method, the basestability of the DEN-2 PDK 53 parental vaccine vector, stored inphosphate buffered saline (PBS), at different temperatures wasdetermined (Table 1). In one example, 1×10⁴ pfu of DEN-2 PDK 53 virus ina total volume of 0.5 ml PBS was incubated, in 2 ml screw capped vialsat either 4° C., room temperature (˜21° C.) or 37° C. After 24 hours ofincubation viral titer and activity was determined by a Neutral Redagarose overlay plaque titration assay in Vero cells. As illustrated inTable 1, incubation of DEN-2 PDK 53 in PBS at 4° C. results in anaverage four-fold decrease in viral titer and complete loss in viralactivity when incubated at 37° C. for the same period. These resultsdemonstrate the relatively poor stability of the DEN-2 PDK 53 flavivirusin the absence of stabilizing excipients.

TABLE 1 Stability of Den-2 PDK53 virus stored for 24 hours at differenttemperatures. Percentage Viral Temperature Formulation Titer Loss  4° C.PBS 75 ~21° C.  PBS 78 37° C. PBS 100

Example 2 Stabilizing Effects of Compositions

In certain exemplary compositions, pharmaceutically acceptableexcipients contemplated herein that aid in thermal stability of liveviral vaccines are known in the art. In one exemplary method, PBS wasused as a base composition to assess the stabilizing effects ofdifferent excipients. In these examples, a stock solution of eachexcipient was made in PBS and the pH adjusted to approximately 7.1 withNaOH, except for chitosan where the pH of the stock solution wasadjusted to approximately 6.8. Excipients were diluted in PBS to thefinal concentrations indicated (w/v) (Table 2). In accordance with thismethod, 1×10⁴ pfu of DEN-2 PDK 53 virus, in serum-free medium, was addedto 0.5 ml of each composition and stored at 37° C. for 24 hours.Following incubation, viral activity and titer was determined by plaquetitration in Vero cells, as described above. As illustrated in Table 2,the stabilizing effects of compositions including a single excipient, atvarious concentrations comparable to previous experimental examples, wasminimal. However, some excipients for example, trehalose and recombinanthuman serum albumin (rHSA), were more effective than others atstabilizing DEN-2 PDK 53 virus at 37° C. Results of the studyrepresented in Table 2 also revealed that increased stabilizing effectsof several excipients, including rHSA and trehalose, can be obtainedwithin certain ranges of concentrations of these excipients. In thisparticular example, trehalose was more effective at concentrations above15% (w/v) and Pluronic F127® (poloxamer 407) at concentrations between0.5 and 3%.

TABLE 2 Effects of different excipients on DEN-2 PDK53 stability whenstored at 37° C. for 24 hours Percentage Viral Formulation Titer LossPBS 100.0 10% Sucrose 99.9 15% Sucrose 98.3 20% Sucrose 96.4 25% Sucrose93.4 2% Trehalose 98.3 5% Trehalose 97.0 10% Trehalose 93.3 15%Trehalose 83.3 2% Mannitol 100.0 5% Mannitol 100.0 10% Mannitol 99.8 15%Mannitol 86.7 5% Sorbitol 100 10% Sorbitol 99.9 15% Sorbitol 99.9 1%Polyvinyl Pyrrolidone 100.0 5% Polyvinyl Pyrrolidone 100.0 10% PolyvinylPyrrolidone 100.0 0.2% F127 99.6 0.5% F127 99.6 1% F127 99.5 2% F12799.5 10% F127 99.9 0.1% rHSA 91.2 0.5% rHSA 95.0 1.0% rHSA 89.0 3.0%rHSA 89.0 5.0% rHSA 97.5 0.05% Chitosan 99.0 0.1% Chitosan 99.0

Example 3 Stabilizing Effects of Compositions Including SpecificCombinations of Excipients

In the following illustrative procedure, compositions including multipleexcipients in differing combinations and concentrations were tested forstabilizing effects on the parental DEN-2 PDK 53 flaviviral vaccine.Excipients were diluted to the indicated final concentrations in PBSfrom stock solutions as described in Example 2. 1×10⁴ pfu of DEN-2 PDK53 vaccine virus was incubated at 37° C. in 0.5 ml of each compositionfor 21 hours (FIG. 1) or over a 48 hour period (FIG. 2). At thespecified time intervals viral titer and activity was determined by aplaque titration assay as described in example 1. FIG. 1 representsexemplary results of this demonstration expressed as percentage of viraltiter remaining after incubation, relative to input, and as log₁₀ titerloss in FIG. 2. Analysis of different combinations of excipients, inthis particular illustration, revealed that formulations consisting of asaccharide, a pluronic co-polymer non-ionic surfactant and a proteinwere optimal at improving DEN-2 PDK 53 stability at 37° C. Formulationsincluding trehalose, copolymer poloxamer 407 (Pluronic F127®) and rHSAhad the greatest stabilizing effects. Unexpectedly, the combinedstabilizing effect of these three excipients was much greater than thesum of that observed with each individual component suggesting synergismbetween the components. Improved thermal stability of the DEN-2 PDK 53flavivirus was obtained through the synergistic activities of thecombination of trehalose, copolymer poloxamer 407 (Pluronic F127) andrHSA could not have been anticipated based on prior art examples. FIGS.1 and 2 also illustrate that the stabilizing effect of thetrehalose/copolymer poloxamer 407 (Pluronic F127®)/rHSA mixture wasfurther enhanced by the addition of 0.05% chitosan. FIG. 2 shows thatthe rate of viral inactivation when stored over a 48 hour period at 37°C. is significantly reduced by compositions containing trehalose,copolymer poloxamer 407 (Pluronic F127®) and rHSA. Examples in the artsuggest that the stability of flaviviruses can be enhanced byformulations containing Ca²⁺ and Mg²⁺ divalent cations. However, asrepresented in FIGS. 1 and 2, the addition of Ca²⁺ (0.0009M) and Mg²⁺(0.0005M) to a formulation confers no additional stabilizing benefits.The results from FIG. 2 suggest that addition of divalent cations mayhave a negative impact to long term liquid phase viral stability in thecontext of particular embodiments.

In one exemplary method, a composition including trehalose, poloxamer407 (Pluronic F127®) and rHSA was assessed for its stabilizingproperties with multiple flaviviruses. The stability of chimeric DEN-2flaviviruses expressing the membrane and envelope proteins from eitherWest Nile (DEN-2/WN), Dengue 1 (DEN-2/D1), Dengue 3 (DEN-2/D3, or Dengue4 (DEN-2/D4) viruses was determined as described for Example 1.Illustrative results in Table 3 reveal greatly improved liquid phasestability of all the chimeric flaviviruses when stored in a compositionincluding trehalose, poloxamer 407 (Pluronic F127®) and rHSA. Thedifferent chimeras express different envelope and membrane proteins fromfive serologically distinct flaviviruses. In addition, West Nile virusand the dengue viruses are significantly divergent. This result suggeststhat compositions herein may be useful for liquid phase stabilization ofdiverse members of the family of Flaviviradae as well as other virusfamilies. The ability to stabilize flaviviruses at room temperature(˜21° C.) and at 4° C. was examined by representative procedures asoutlined for Example 1. The exemplary results, illustrated in Table 4,reveal that a composition including trehalose, poloxamer 407 (PluronicF127®) and rHSA effectively preserves viral activity for 7 days at 21°C. and for 48 days at 4° C.

TABLE 3 Stability of different chimeric flaviviruses stored at 37° C.for 21 hours in PBS or a composition (F1) including 15% trehalose, 2%poloxamer 407 (Pluronic F1 27 ®) and 1% rHSA. % Viral Titer VirusFormulation Remaining DEN-2/WN PBS 2 F1 45 DEN-2/D1 PBS 0.2 F1 22DEN-2/D3 PBS 0.3 F1 30 DEN-2/D4 PBS 1 F1 28

TABLE 4 Stability of flaviviruses stored at different temperatures for 7or 48 days in PBS or a composition (F1) including 15% trehalose, 2%copolymer poloxamer 407 (Pluronic F127 ®) and 1% rHSA. Percentage ViralTiter Remaining 7 48 Virus Temperature Formulation days days DEN-2PDK-53 21° C. PBS 0 0 21° C. F1 100 0  4° C. PBS 0 0  4° C. F1 100 100DEN-2/WN 21° C. PBS 0 0 21° C. F1 100 0  4° C. PBS 0 0  4° C. F1 100 100

Example 4 Use of Alternate Components

Another exemplary method was used to compare the stabilizing effects ofbovine serum albumin (BSA) and, gelatin, to that of rHSA and ofdifferent pluronic co-polymers. DEN-2 PDK 53 viral stability assays wereconducted as outlined previously for Examples 1 and 2. The previousexamples suggested that formulations including trehalose, poloxamer 407(Pluronic F127) and rHSA optimally improved the thermal stability of theDEN-2 PDK 53 parental vaccine virus. As shown by example in FIG. 3, thestabilizing effects of bovine serum albumin are comparable to those ofrHSA either alone or in combination with trehalose and poloxamer 407(Pluronic F127®). FIG. 3 also demonstrates that as isolated excipients,gelatin is comparable to rHSA in stabilizing DEN-2 PDK 53 at 37° C.However in this exemplary method, unlike BSA, gelatin does not appear tobe an effective substitute for rHSA in compositions also containingtrehalose and poloxamer 407 (Pluronic F127®). Thus, while proteins otherthan rHSA may be used in combination with trehalose and poloxamer 407(Pluronic F127®) to aid in stabilization of flaviviral vaccines, the useof a serum albumin or closely related proteins is more suitable inaccordance with this exemplary method. In addition, FIG. 3 illustratesthat, as isolated excipients, the polymer Pluronic P123® (poloxamer 403)is comparable to poloxamer 407 (Pluronic F127®) in its ability tostabilize the DEN-2 PDK-53 virus. However, in this exemplary method,poloxamer 403 (Pluronic P123®) does not appear to be an effectivesubstitute for poloxamer 407 (Pluronic F127®) in compositions alsocontaining trehalose and serum albumin. As exemplified in FIG. 4,compositions containing trehalose, rHSA and other commonly usedpharmaceutical surfactants such as Polysorbate 20 (Tween 20), instead ofa pluronic co-polymer, are not effective in stabilizing DEN-2 PDK 53relative to formulations containing a pluronic co-polymer. Theseexemplary methods suggest better stabilizing efficiencies offormulations containing distinct high molecular weight pluronicco-polymer surfactants.

Exemplary data is further illustrated in FIG. 4. FIG. 4. representsstability of the DEN-2 PDK 53 virus in compositions containing differentsurfactants. DEN-2 PDK 53 was stored at 37° C. for 23 hours in eachformulation. Surfactants evaluated in this example includen-octyl-β-D-glucopyranoside (β-OG), Polysorbate 20 (P 20), Polysorbate80 (P 80) and copolymer poloxamer 407 (Pluronic F127®; (F)). Otherformulation components include trehalose (T) and rHSA (A). Values areexpressed as a percentage of the viral titer remaining after incubationrelative to the input titer.

Example 5 Comparison of the Stabilizing Effects of DifferentCompositions

The stabilizing properties of one exemplary composition were compared tothat of compositions known in the art. A stabilizing composition forlive flaviviral vaccines, disclosed in the art (U.S. Pat. No.4,500,512), includes 4% lactose, 2% sorbitol, 0.1 g/L CaCl₂, 0.076 MgSO₄and amino acids on the order of 0.0005M to 0.05M in PBS. Anothercomposition reported by Adebayo et al (1998) consists of 10% sucrose, 5%lactalbumin, 0.1 g/L CaCl₂, and 0.076 g/L MgSO₄. In one exemplarymethod, stabilizing properties of these formulations were compared to aparticular embodiment herein. In one example composition, F1, thiscomposition includes 15% trehalose, 2% copolymer poloxamer 407 (PluronicF127®) and 1% recombinant HSA. F2 is the formulation of U.S. Pat. No.4,500,512 without amino acids and F3 is the same formulation with theamino acids histidine and alanine. F4 is the composition of Adebayo, etal. 1×10⁴ pfu of DEN-2 PDK 53 vaccine virus were incubated at 37° C. in0.5 ml of each composition for 23 hours, after which viral activity andtiter was assayed as described in Example 1. As exemplified in FIG. 5,some embodiments, for example formulation F1, represents a significantimprovement over those previously described compositions. In the exampleshown, virtually no viral activity was recovered after storage in theformulations known in the art (formulations F3 and F4), whereas upwardsof 50% of the initial viral titer was recovered after storage in acomposition disclosed herein. These results reveal that previousformulations are ineffective at promoting live viral vaccine stabilityduring liquid phase storage.

Example 6

Preservation of Viral Activity after Multiple Freeze-Thaws

In one exemplary method, the ability of select compositions to preserveviral activity after freeze-thaw cycles was demonstrated. 1×10⁴ pfu ofDEN-2 PDK 53 vaccine virus was suspended in 0.5 ml of each compositionin screw cap vials. For the first freeze-thaw cycle vials were frozen at−80° C. for 24 hours and thawed rapidly at 37° C. This was immediatelyfollowed by a second freeze-thaw cycle where the vials were frozen at−80° C. for 1 hour and thawed rapidly at 37° C. Viral titer and activitywas then assessed by a plaque titration assay as described in Example 1.As illustrated in FIG. 6, particular compositions that includetrehalose, copolymer poloxamer 407 (Pluronic F127®) and rHSA effectivelypreserved full viral activity through two freeze-thaw cycles.Additionally, compositions including these three excipients were moreeffective than those containing just a single excipient. The results ofthis particular illustrative experiment suggest the compositions andmethods disclosed herein are an effective cryoprotectant for flaviviralvaccines and may facilitate viral preservation during freeze-drying,spray-drying, or other dehydration techniques.

Example 7 Stabilization of Other Live, Attenuated Viruses.

Examples illustrated previously reveal effective liquid phasestabilization of several live, attenuated flaviviruses in compositionsincluding trehalose, poloxamer 407 (Pluronic F127®) and rHSA. It isanticipated that embodiments disclosed herein may also be effective atstabilizing other live, attenuated viruses. For example, a formulationincluding trehalose, poloxamer 407 (Pluronic F127®) and rHSA may be usedto stabilize live attenuated measles virus, an attenuated sindbis virus,an attenuated influenza virus, a recombinant, attenuated adenovirus or arecombinant, attenuated vaccinia virus. In one exemplary method, thesenon-flaviviral viruses can be suspended and maintained in liquid phase,in a composition including trehalose, poloxamer 407 (PluronicF127®), andrHSA directly after harvesting from cell culture. In anotherillustrative method, non-flaviviral viruses can be suspended in acomposition prior to, or subsequent to, freeze or spray-drying.Statistically improved viral stability may demonstrate that theformulation of this embodiment is applicable to other attenuated viralvaccines outside of the Flavivirus family. Those skilled in the artrecognize that application may then be extended to other live,attenuated viruses.

Example 8 Safety and In Vivo Immunogenicity.

Molecular interactions between excipients and molecular or cellularcomponents may serve, not only to enhance stability of viral vaccines,but also to cause increased cell or tissue damage in vivo. Theformulations may decrease the immunogenicity of these viral vaccines inlive animals. In this example, it is demonstrated that exemplarycompositions are safe after subcutaneous injection and are essentiallyimmunologically inert. Four different exemplary compositions wereselected for testing in mice as follows.

Formulation 1: 15% Trehalose, 2% Pluronic F-127 (poloxamer 407), 1% rHSAFormulation 2: 15% Trehalose, 2% Pluronic F-127® (poloxamer 407), 1%rHSA, 1 mM CaCl₂/0.5 mM MgSO₄Formulation 3: 15% Trehalose, 2% Pluronic F-127® (poloxamer 407), 1%rHSA, 0.5% chitosanFormulation 4: 22.5% Trehalose, 3% Pluronic F-127® (poloxamer 407), 1.5%rHSA

Formulation 5: PBS

In certain methods described herein, groups of 8 or 9 NIH Swiss micewere immunized by subcutaneous injection with 1×10⁵ pfu of a formulatedDEN-2 PDK-53/WN recombinant flavivirus vaccine at day 0 (d0), wereboosted with the same formulated vaccine at d29 and were then challengedwith 10³ pfu on a pathogenic West Nile strain (NY99) on d45. Controlmice (four groups of 8) received formulations 1-4 alone with no virus.No adverse events after administration in any of the immunized mice wereobserved. Thus, in this example, no apparent adverse events are causedby the exemplary formulations with or without vaccine virus. Sera werecollected prior to immunization at d0, prior to boost at d28, prior tochallenge at d44 and post-challenge at d75. West Nile neutralizingantibody titers in the sera were determined by plaque reductionneutralization test (PRNT). The results of the study are represented inTable 5.

TABLE 5 Neutralizing antibody and protection induced by formulatedDEN2/WN vaccines Post-prime Post-boost Post-Challenge (d 28) (d 44) (d75) Formulation Number GMT¹ % SC² GMT % SC GMT % SC Survival % Survival1 8 30 87.5 123 100 761 100 8/8 100 2 8 10 62.5 226 100 830 100 8/8 1003 8 40 100 123 100 1810 100 8/8 100 4 9 10 66.7 137 100 1660 100 8/988.9 5 9 10 66.7 109 100 1742 100 9/9 100 Controls 32 1 0 1 0 1280 100 7/32 21.9 ¹GMT = geometric mean titer; titers of <10 were arbitrarilyassigned a value of 1. ²% SC = percentage of animals that sero-convertedwith PRNT titers >10.

A majority of the animals receiving the DEN-2/WN vaccine sero-convertedafter the first dose regardless of whether no formulation (Formulation5) or one of the exemplary formulations (Formulations 1-4) was used. Inaddition, all of the vaccinated animals sero-converted after the boosteradministration. Geometric mean PRNT titers (GMT) demonstrate fewdifferences between the vaccine groups. Titers were low after theprimary immunization, increased 3-10 fold after the boost and thenshowed a dramatic anamnestic response upon challenge. 100% of all thevaccinated animals survived challenge, again independent of vaccineformulation. Only 22% of the control animals survived; those that didsurvive showed evidence of potent neutralizing antibody responses afterchallenge. One advantage is that this example demonstrates that theexemplary formulations do not reduce the ability of an exemplaryrecombinant DEN-2/WN vaccine to prevent West Nile disease in a mice.

Example 9

In another example, liquid compositions were used containing trehalose,rHSA and poloxamer 407 (Pluronic F127®) to stabilize a West Nilechimeric flavivirus stored for various periods at either 25° C. or 4° C.1×10⁴ pfu of chimeric DEN-2/WN vaccine virus were incubated at eachtemperature and viral activity was assessed at one or two week intervalsas described in Example 1. As illustrated in FIGS. 7 and 8, formulationscontaining trehalose, rHSA and poloxamer 407 (Pluronic F127®)significantly improved the thermal stability of the DEN-2/WN vaccinevirus during storage at 25° C. and 4° C., respectively. At 25° C. lossof viral activity was less than one log over 7 days. At 4° C. viralinactivation was negligible for periods up to 12 weeks when stored inexemplary formulations including trehalose, poloxamer 407 (PluronicF127®) and rHSA.

Example 10

In another exemplary method, stabilizing effects of compositions weredemonstrated including trehalose, rHSA and a pluronic co-polymer withdehydrated DEN-2 PDK 53 vaccines. 1×10⁴ pfu of DEN-2 PDK 53 vaccinevirus formulated in accordance with procedures disclosed herein.Formulated vaccines were placed in serum vials and subjected toconventional lyophilzation procedures. Dried vaccines were stopperedunder vacuum, stored at either 37° C. or 4° C. for 14 days followed byreconstitution of the vaccine to its original liquid volume by additionof sterile water. Viral activity of the reconstituted vaccine wasassessed as outlined earlier. At 37° C., in the presence of compositionscontaining trehalose, rHSA and a pluronic co-polymer formulated inphosphate buffered saline, an average viral titer loss of 1 log wasobserved (FIG. 9). No loss in viral activity was observed for formulateddehydrated DEN-2 PDK 53 viral vaccines stored at 4° C. for 14 days.These results demonstrate effective preservation of a dehydrated viralvaccine utilizing compositions disclosed herein.

FIG. 9. represents stability of lyophilized DEN-2 PDK 53 at differenttemperatures. Log titer loss of formulated lyophilized DEN-2 PDK 53vaccine virus following incubation at 37° C. or 4° C. for 2 weeks asindicated. Formulations F1 (15% trehalose, 2% poloxamer 407 (PluronicF127®), 1% rHSA) and F2 (15% trehalose, 2% poloxamer 407 (PluronicF127®), 0.01% rHSA) were formulated in phosphate buffered saline.Formulation F3 (15% trehalose, 2% poloxamer 407 (Pluronic F127®), 0.01%rHSA) was formulated in 10 mM Tris base.

All of the COMPOSITIONS and METHODS disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods have been described interms of preferred embodiments, it is apparent to those of skill in theart that variations maybe applied to the COMPOSITIONS and METHODS and inthe steps or in the sequence of steps of the methods described hereinwithout departing from the concept, spirit and scope herein. Morespecifically, certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept as defined bythe appended claims.

All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety.

1-29. (canceled)
 30. A virus composition comprising: one or more live,attenuated flaviviruses; and a stabilizing composition comprising serumalbumin and trehalose, wherein the one or more live, attenuatedflaviviruses consist of one or more Dengue viruses; wherein the serumalbumin is serum albumin from a vertebrate species, and is present at aconcentration of 0.001% to 3.0% (w/v); wherein the trehalose is presentat a concentration of at least 5% (w/v); and wherein the composition iscapable of reducing the inactivation of the one or more live, attenuatedDengue viruses.
 31. The virus composition according to claim 30, whereinthe trehalose is present at a concentration of a 5% to 50% (w/v). 32.The virus composition according to claim 30, wherein the trehalose ispresent at a concentration of at least 10% (w/v).
 33. The viruscomposition according to claim 30, wherein the trehalose is present at aconcentration of 5% to 15% (w/v).
 34. The virus composition according toclaim 33, wherein the trehalose is present at a concentration of 10% to15% (w/v).
 35. The virus composition of claim 30, wherein the serumalbumin from a vertebrate species is from a human, a canine, or a bovinesource.
 36. The virus composition of claim 35, wherein the serum albuminfrom a vertebrate species is from a human source.
 37. The viruscomposition of claim 30, wherein the serum albumin is recombinantalbumin.
 38. The virus composition of claim 30, wherein the viruscomposition further comprises one or more poly(ethylene oxide) andpoly(propylene oxide) (EO-PO) block copolymers, and the one or moreEO-PO block copolymers include poloxamer
 407. 39. A method fordecreasing inactivation of one or more live, attenuated Dengue virusescomprising: combining one or more live, attenuated Dengue viruses with astabilizing composition comprising serum albumin and trehalose, whereinthe serum albumin is serum albumin from a vertebrate species, and ispresent at a concentration of 0.001% to 3.0% (w/v); wherein thetrehalose is present at a concentration of at least 5% (w/v), andwherein the composition is capable of reducing the inactivation of theone or more live, attenuated Dengue viruses.
 40. The method according toclaim 39, wherein the trehalose is present at a concentration of a 5% to50% (w/v).
 41. The method according to claim 39, wherein the trehaloseis present at a concentration of at least 10% (w/v).
 42. The methodaccording to claim 39, wherein the trehalose is present at aconcentration of 5% to 15% (w/v).
 43. The method according to claim 42,wherein the trehalose is present at a concentration of 10% to 15% (w/v).44. The method of claim 39, further comprising partially or whollydehydrating the combination.
 45. The method of claim 39, furthercomprising partially or wholly re-hydrating the composition prior toadministration.
 46. The method of any one of claim 39, wherein thecomposition increases the shelf life of an aqueous Dengue viruscomposition.
 47. The method of claim 39, wherein the compositiondecreases inactivation of an aqueous live, attenuated Dengue virus for24 hours or greater.
 48. The method of claim 39, wherein the compositiondecreases inactivation of an aqueous live, attenuated Dengue virusduring one or more freeze and thaw cycles.
 49. The method of claim 39,wherein the composition comprises one or more live, attenuated Dengueviruses and is administered to a subject to reduce the onset of a healthcondition caused by the Dengue viruses.
 50. A kit for decreasing theinactivation of Dengue viruses comprising: at least one container; and acomposition comprising one or more live, attenuated Dengue viruses; anda stabilizing composition comprising: serum albumin and trehalose,wherein the serum albumin is serum albumin from a vertebrate species,and is present at a concentration of 0.001% to 3.0% (w/v); wherein thetrehalose is present at a concentration of at least 5% (w/v); andwherein the composition is capable of reducing the inactivation of theone or more live, attenuated Dengue viruses.
 51. The kit according toclaim 50, wherein the trehalose is present at a concentration of a 5% to50% (w/v).
 52. The kit according to claim 50, wherein the trehalose ispresent at a concentration of at least 10% (w/v).
 53. The kit accordingto claim 50, wherein the trehalose is present at a concentration of 5%to 15% (w/v).
 54. The kit according to claim 50, wherein the trehaloseis present at a concentration of 10% to 15% (w/v).
 55. The kit of claim50, wherein the serum albumin is human serum albumin.